Paracrine signals from mesenchymal feeder cells and regulating expansion and differentiation of hepatic progenitors using same

ABSTRACT

A method is provided for controlling the survival, proliferation, and/or differentiation of hepatic progenitors in vitro by using specific types of mesenchymal feeder cells or one of more of the paracrine signals produced by those feeders.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority from U.S. Provisional Application No.60/944,435, filed Jun. 15, 2007, incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to the ex vivo propagationand/or differentiation of hepatic progenitor cells. More particularly,the present invention relates to the identification and selection ofsoluble and insoluble paracrine signals derived from mesenchymal cellsand their application in regulating expansion and/or differentiation ofhepatic progenitor cells, including hepatic stem cells, in vitro.

BACKGROUND OF THE INVENTION

Hepatic stem cells and their progeny (e.g., hepatoblasts and committedprogenitors) have considerable expansion potential. For this reason,these cell populations are desirable candidates for cell therapies,including bioartificial livers or cell transplantation. Despite thispromise, however, the full potential of liver cell therapy remains to berealized.

The in vitro propagation of hepatic stem cells and their progeny hasproven to be challenging, in part, because in vitro culture conditionsare not always optimal for transition from the laboratory bench to theclinic. For example, some culture conditions are not good for survival,can greatly retard cell division, or can promote cell differentiationtowards undesired fates. As well, some culture conditions require theaddition of factors (e.g., serum) that can introduce contaminants andthereby limit their application in treating humans.

Maintenance of normal cells, especially progenitors, requires feeders ofmesenchymal companion cells, known to provide paracrine signals criticalfor survival and function of the progenitors. There is a need toidentify categories of mesenchymal cell feeders and then to use them asmodels to identify their paracrine signals, extracellular matrixcomponents and soluble signals, that mediate expansion, lineagerestriction towards specific fates, or differentiation of hepaticprogenitors towards their adult fates of biliary epithelia andhepatocytes. Defining the signals enables one to use the signals ontheir own in the proper combinations and without the feeders to elicitthe desired biological responses from the hepatic progenitors and thatincludes survival, expansion, lineage restriction towards a fate, andfull differentiation to mature liver cells. Thus, there is a need forculture conditions that are defined so as to obviate the heretoforerequirement of feeder cells.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, a method of propagatinghepatic stem cells in vitro without inducing their differentiation isprovided comprising: culturing a population of isolated hepatic stemcells in serum-free culture media and on a layer of matrix componentsselected from the group consisting of hyaluronans, other unsulfated orpoorly sulfated glycosaminoglycans (GAGs), unsulfated or poorly sulfatedproteoglycans, embryonic collagens (e.g., type III) and embryonic basaladhesion molecules, and combinations thereof, in which the layer isessentially free of mature collagens (e.g., type I), and in which theculturing propagates the hepatic stem cells without inducing theirdifferentiation.

Any or all of the matrix components may be supplied by angioblast feedercells, quiescent hepatic stellate feeder cells, HUVEC feeder cells, or acombination thereof. The basal adhesion molecules may comprise isoformsof laminin found predominantly in fetal tissues and the GAGs, other thanhyaluronans, may be forms of chondroitin sulfates. The hepatic stemcells may be human and obtained from fetal, neonatal, pediatric or adultliver. The laminin may be supplied at a concentration between about 0.1to about 2 μg/cm², preferably at a concentration of about 1 μg/cm².Similarly, the type III or IV collagens can be, individually, at aconcentration between about 0.1 to about 15 μg/cm².

In another embodiment of the present invention, a method ofdifferentiating hepatic stem cells in vitro to hepatoblasts is providedcomprising: culturing a population of isolated hepatic stem cells inserum-free culture media and on a layer of matrix components selectedfrom the group consisting of embryonic collagens, basal adhesionmolecules, CS-PGs, and combinations thereof, in which the layer isessentially free of mature collagens, and in which the culturingpropagates the hepatic stem cells without inducing theirdifferentiation.

Any or all of the matrix components may be supplied by activatedendothilia, activated hepatic stellate feeder cells, or both. Theembryonic collagen can be a type IV collagen and the basal adhesionmolecules may comprise fetal isoforms of laminin, supplied at aconcentration between about 0.1 to about 2 μg/cm², preferably at aconcentration of about 1 μg/cm². In some embodiments, the layer furthercomprises hyaluronans. The hepatic stem cells can be obtained fromfetal, neonatal, pediatric or adult liver, and preferably from humans.

In yet another embodiment of the present invention, a method ofdifferentiating hepatic stem cells or hepatoblasts in vitro intocommitted hepatocyte or biliary progenitors and progeny thereof isprovided comprising: culturing a population of isolated hepatic stemcells in serum-free culture media and on a layer of matrix componentsselected from the group consisting of sulfated proteoglycans, maturecollagens, fibronectin, and combinations thereof, and in which theculturing induces the differentiation of the hepatic stem cells orhepatoblasts into committed hepatic or biliary progenitors and progenythereof. Any or all of the matrix components may be supplied by stromalfeeder cells, activated hepatic stellate feeder cells, myofibroblastfeeder cells, or combinations thereof. In some embodiments, the layer issubstantially free of hyaluronans and the sulfated proteoglycans can beheparan sulfate-PG or heparin-PG, or both.

In still yet another embodiment of the present invention, a containerfor propagation of hepatic progenitors or differentiating them isprovided. The containers comprise a layer of matrix components selectedfrom the group consisting of hyaluronans, other unsulfated or poorlysulfated glycosaminoglycans (GAGs), unsulfated or poorly sulfatedproteoglycans, embryonic collagens and embryonic basal adhesionmolecules, and combinations thereof; wherein the layer is essentiallyfree of mature collagens; and wherein the layer of matrix componentssubstantially coats at least one surface of the container.

Alternatively, the layer may comprise matrix components selected fromthe group consisting of embryonic collagens, basal adhesion molecules,CS-PGs, and combinations thereof, wherein the layer is essentially freeof mature collagens; and wherein the layer of matrix componentssubstantially coats at least one surface of the container. Finally, thelayer may comprise matrix components selected from the group consistingof sulfated proteoglycans, mature collagens, fibronectin, andcombinations thereof, wherein the layer of matrix componentssubstantially coats at least one surface of the container. The containermay be a tissue culture plate, a bioreactor, a lab cell or a lab chip.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains multiple figures executed incolor. Copies of this patent or patent application publication withcolor figures will be provided by the Office upon request and withpayment of the necessary fee.

FIG. 1 shows a colony of hHpSCs and human hepatoblasts in culture: BothhHpSC colony (A) and human hepatoblasts (B) expressed EpCAM (shown ingreen). NCAM (shown in red, A) was expressed along the peripheral regionand the center of the colony. Hepatoblasts strongly expressedalpha-fetoprotein (AFP, shown in red, B). Both cells were stained withDAPI (blue). Scale bar, 100 μm.

FIG. 2 shows that cells surrounding a colony of hHpSCs are αSMA+hHpSTCs:A typical human hepatic stem cell colony (A) is positive for NCAM insidethe colony (B, D) and for αSMA in the companion cells at the edge ofcolony (C, D). Magnification, 10×.

FIG. 3 shows a primary culture of hHpSCs with angioblasts: KDR+ selectedcells cultured in EGM-2 for 7 days expressed vWF (green) (A and B).CD31+ selected cells cultured in EGM-2 for 4 days expressed both vWF(green) and CD31 (red) (C). Scale bar, 100 μm. Angioblasts associatedwith hHpSC colony in culture (D). Magnification, 10×.

FIG. 4 compares quiescent with activated hHpSTCs: Quiescent hHpSTCsexpress low levels of desmin, αSMA+, CD146, type I collagen, and othermatrix molecules (fibronectin, proteoglycans). Injury processes—forexample, exposure to serum or to certain factors (e.g., PDGF andTGF-B1)—cause hHpSTCs to activate and to transition tomyofibroblast-like stromal cells and elevate production of αSMA andmatrix components, and to release various growth factors such as HGF.Shown is a colony of hHpSCs encircled by mesenchymal companion cells(angioblasts and quiescent hHpSTCs) expressing low levels of CD146. Onthe same plate, an adjacent colony is shown with the mesenchymalcompanion cells that have undergone activation resulting in high levelsof CD146.

FIG. 5 shows the morphology and immunohistochemistry of differentfeeders: A: hMSCs. B: hUVECs. C-D: human fetal liver-derived feedercells on days 4 (C) and 7 (D). E-H: day-11 culture of magneticallyimmunoselected KDR+ cells (E-F) and supernatant cells depleted offibroblasts (G-H) in serum-free conditions were positive for αSMA (F andH). Magnification, 10×.

FIG. 6 shows immunohistochemistry on fibroblast-depleted supernatantcells cultured in EGM-2 medium for 8 days: Cells were positive fordesmin (B and H), αSMA (I), laminin (C), fibronectin (F), collagen typesI (L) and IV (E), and negative for endothelial marker vWF (K). Note thatmore cells express αSMA than desmin. Phase contrast image for eachdouble staining is shown (A, D, G, and J). Bar, 50 μm.

FIG. 7 shows some effects of hHpSCs co-cultured with different feeders.A-F: hHpSCs cultured alone (A), co-cultured with hUVECs (B), hMSCs (C),or human fetal liver-derived feeders (D). Magnification, 10×.

FIG. 8 shows human hHpSCs co-cultured with αSMA+ supernatant cellsderived from human fetal livers, from which fibroblasts were depleted.Use of these feeders resulted in lineage restriction to hepatoblasts.Immunohistochemistry for human AFP on human hepatic stem cell colonies(A and B) and on co-culture of hHpSCs and human fetal liver-derivedfeeders (C and D) at day 8. Magnification, 10×.

FIG. 9 shows normalized mRNA expression for mRNAs encoding matrixmolecules: fold changes of mRNA expression levels in each cell type werenormalized to ribosomal RNA (18S) content of the same cell type.

FIG. 10 shows the behavior of hHpSCs on substrata of purified matrixcomponents: hHpSCs maintain stem cell characteristics on plastic or ontype III collagen (A and B). hHpSCs lineage restrict to hepatoblastswhen cultured on top of type IV collagen or on laminin (C and D). hHpSCsfurther differentiate into mature hepatocytes when cultured atop type Icollagen. Higher magnification for D.

FIG. 11 provides a summary of the changes in matrix chemistry and matrixreceptors in the hHpSCs and their mesenchymal cell partners duringdifferentiation.

FIG. 12 provides a comparison of human cytokines produced in co-cultureand in human fetal liver cell culture. Concentration (pg/ml) of humancytokines produced in human fetal liver cell single culture and inco-culture of STO feeder cells and human fetal liver cells at low (top)and high (bottom) levels.

FIG. 13 provides a comparison of mouse cytokines produced in co-cultureand in STO feeder cell culture. Concentration (pg/ml) of mouse cytokinesproduced in STO feeder cell single culture and in co-culture of STOfeeder cells and human fetal liver cells at low (top) and high (bottom)levels.

FIG. 14 shows the cytokine effects on colony formation of rter6 cells.Colony number (top) and area (in pixels; bottom) of rat hepaticprogenitor (rter6) cells in hormonally defined medium (HDM) with orwithout cytokine are shown.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, extracellular matrixcomponents have been identified, which facilitate the attachment,survival and ex vivo proliferation and other matrix components thatelicit the differentiation of hepatic stem cells and their progeny. Theterm “hepatic progenitors,” as used herein, is broadly defined toencompass both hepatic stem cells and their progeny. “Progeny” mayinclude both hepatic stem cells or hepatoblasts, both pluripotentprogenitors thereof, and committed progenitors that can differentiateinto only one lineage leading to particular mature cell type (e.g., ahepatocyte).

“Clonogenic expansion” refers to the growth property of cells that canexpand from a single cell and be subcultured and expanded repeatedlywith retention of the phenotype of the parental cell. “Colony formation”refers to the property of diploid parenchymal cells that can undergo alimited number of cell divisions (typically 5-7 cell divisions) within aweek or two and involves cells with limited ability to undergosubculture or passaging. “Pluripotent” signifies cells that can formdaughter cells of more than one fate; “unipotent” or “committedprogenitors” are cells that have a single adult fate.

Hepatic stem cells (HpSCs) are pluripotent cells found in the ductalplates (also called limiting plates) in fetal and neonatal livers and inthe Canals of Hering in pediatric and adult livers and showing evidenceof self-replication with expression of telomerase and being capable offorming mature liver cells when transplanted. These cells are EpCAM+,NCAM+, ALB+, CK8/18+, CK19+, CD133/1+, and are negative for allhemopoietic markers tested (e.g., CD34, CD38, CD45, CD14), mesenchymalcell markers (CD146, VEGFr, CD31) and for expression of P450s oralpha-fetoprotein. The HpSCs have been found to give rise tohepatoblasts and to committed (unipotent) progenitors.

Hepatoblasts (HBs) are bipotent cells found throughout the parenchyma offetal and neonatal livers and as single cells or small aggregates ofcells tethered to the ends of the Canals of Hering. HBs derive from theHpSCs. HBs share many antigens present on HpSCs but with importantdistinctions. For example, HBs do not express NCAM but rather ICAM1 andthey express significant amounts of alpha-fetoprotein and fetal forms ofP450s. These HBs give rise to the unipotent progenitors, the committedhepatocytic and biliary progenitors.

Committed hepatic progenitors are unipotent progenitors of either thehepatocytic or biliary lineages. Their antigenic profile overlaps withthat of the HBs; however, biliary committed progenitors express CK19 butnot AFP or ALB, whereas the hepatocytic committed progenitors expressAFP and ALB but not CK19. Committed biliary progenitors derive directlyfrom hepatic stem cells and also from hepatoblasts.

Mesenchymal cells (MCs) include cells at various lineage stages of themany different mesenchymal cell types (listed as the mature cells and,in parentheses, their precursors): including stroma (mesenchymal stemcells), endothelia (angioblasts), stellate cells (stellate cellprecursors), and various hemopoietic cells (hemopoietic stem cells)

While most, if not all, of the discussion and examples of hepaticprogenitors herein will be with reference to human-derived cellpopulations, the teachings herein should not be limited to humans. Infact, one of ordinary skill in the art may be expected to apply theteachings herein to the expansion of hepatic progenitors from mammals,generally (e.g., mice, rats, dogs, etc.). Accordingly, the scope of thepresent invention is intended to include hepatic progenitors of any andall mammals.

It is also noted that hepatic progenitors suitable for in vitropropagation in accordance with the instant invention are not limited tothose isolated or identified by any particular method. By way ofexample, methods for the isolation and identification of the hepaticprogenitors have been described in, for example, U.S. Pat. No. 6,069,005and U.S. patent application Ser. Nos. 09/487,318; 10/135,700; and10/387,547, the disclosures of which are incorporated herein in theirentirety by reference.

Hepatic stem cells and hepatoblasts have characteristic antigenicprofiles and can be isolated by protocols described previously. Forexample, hepatic stem cells and hepatoblasts share numerous antigens(e.g., cytokeratins 8, 18, and 19, albumin, CD133/1, and epithelial celladhesion molecule (“EpCAM”)) and are negative for hemopoietic markers(e.g., glycophorin A, CD34, CD38, CD45, CD14) and mesenchymal cellmarkers (e.g., CD146, CD31, VEGFr or KDR). Alternatively, hepatic stemcells and hepatoblasts can be distinguished from each other by size (thestem cells are 7-9 μm; the hepatoblasts are 10-12 μm), by morphology incultures (the stem cells form dense, morphologically uniform colonies,whereas the hepatoblasts form cord-like structures interspersed by clearchannels, presumptive canaliculi), by distinctions in the pattern ofexpression of certain antigens (EpCAM is expressed throughout thehepatic stem cells but is confined to the cell surface in thehepatoblasts), or by distinct antigenic profiles (N-CAM is present inthe hepatic stem cells, whereas alpha-fetoprotein (AFP) and ICAM1 areexpressed by the hepatoblasts). In fetal and neonatal livers, thehepatic stem cells are in the ductal plates (also called “limitingplates”), whereas the hepatoblasts are the dominant parenchymal cellpopulation (>80%). In pediatric and adult tissues, the hepatic stemcells are present in the Canals of Hering, whereas the hepatoblasts arecells tethered to the ends of the Canals of Hering. The hepatoblastsconsist of small numbers of cells in normal tissue but found in largenumbers (e.g., nodules) in diseased livers (e.g., cirrhosis).

The present invention provides methods to control ex vivo maintenance ofHpSCs, preferably human HpSCs (hHpSCs), in vitro. More specifically, theinventive method enables propagation of HpSCs (1) without inducingdifferentiation (i.e., self-renewal); (2) inducing differentiation(i.e., “lineage restriction”) of the HpSCs to hepatoblasts; or (3)inducing more “extensive” differentiation (e.g., into committedprogenitors) (collectively referred herein a “ex vivo maintenance”). Themethod is enabled, in part, by the selective use of specific types ofmesenchymal feeder cells used in co-cultures. The invention alsoprovides insoluble (e.g., matrix molecules) and soluble (e.g.,cytokines) components that alone or in combination allow for thepropagation of HpSCs, if preferred, in the absence of feeder cells.Table 1 summarizes the insoluble factors discovered relevant to affectthe aforementioned modes of ex vivo maintenance.

TABLE 1 Effects of Feeders/Substratum on Human Hepatic Stem/ProgenitorsMorphology/antigenic profile of cells maintained on feedersSubstratum/Feeder for a week or more Plastic*** hHpSC hUVECs hHpSC hMSCsHepatoblasts Liver Cell KDR+ Cells in hHpSCs Suspensions first weekimmunoselected KDR+ cells Initially mixture of hHpSCs and for: after7-10 days hepatoblasts that transitions rapidly to only hepatoblastsCD31+ Cells Hepatoblasts Depleted of Hepatoblasts stromal cells STOcells Growth-arrested hHpSCs differen- tiating to hepatoblasts andcommitted (unipotent) progenitors Fibronectin Few cells attached; thosethat did rapidly lost viability Laminin Hepatoblasts Type III collagenhHpSCs Type IV collagen Hepatoblasts On surface of Type I collagenGrowth-arrested cells that are a mixture of hepatoblasts and committed(unipotent) progenitors Embedded in Type I collagen Mature hepatocytesAll cultures were in serum-free KM. ***Note: when cells are on cultureplastic, the hHpSC colonies survive only when they are in associationwith companion cells comprised of angioblasts and/or hepatic stellatecell precursors. *Culture Morphology and antigenic profile: hHpSCcolonies are monolayers with cells of uniform morphology, high nucleusto cytoplasmic ratio, ~7-9 μm in diameter, tightly packed and surroundedby companion cells that include angioblasts and hHpSTCs. UniqueAntigenic profile: NCAM+, Claudin 3+, albumin±, AFP−. Hepatoblastsappear as colonies that are more 3-dimensional, with cord-like structureinterspersed by clear channels (bile canaliculi) and with cells that areslightly larger (10-12 μm) in diameter. Unique antigenic profile: ICAM+,Claudin 3−, albumin++, AFP++. Shared antigenic profile between hHpSCsand hepatoblasts: positive for EpCAM, CK 8, 18, and 19, Indian Hedgehogproteins (sonic, Indian), telomerase; negative for hemopoietic markers(CD34, CD45, CD38, glycophorin A), for hepatic stellate cell markers(desmin and αSMA) and for endothelial cell markers (VEGFr, CD31, vWF).

Three distinct classes of feeders have been identified in keeping withthe three modes of ex vivo maintenance outlined above. Co-culture withfeeders of endothelia precursors or angioblasts free of human hepaticstellate cells (hHpSTCs) (or, in the alternative, comprising quiescenthHpSTCs) allow for the expansion of HpSCs without inducing theirdifferentiation. Feeders replete with activated endothelia and hHpSTCslineage restrict HpSCs to hepatoblasts. Finally, feeder cells comprisingmature endothelia or murine stroma (represented by STO cells), leadHpSCs to differentiate into mature parenchymal cells (including, biliaryand hepatocytic cells). It is presently believed that the behavior ofthe co-cultures thus identified parallels that observed during liverdevelopment, which is governed by paracrine signals from mesenchymeadjacent to the epithelium.

Matrix chemistry can be relevant to embryonic development. In oneembodiment of the invention, the present inventors have found thatextracellular matrix components found in or near the liver's stem cellniche, provide for expansion of hepatic progenitors without inducingdifferentiation better than existing technology. As described in U.S.patent application having Ser. No. 11/560,049 filed Nov. 15, 2006—thedisclosure of which is incorporated herein in its entirety byreference—cells cultured on the matrix components, found in abundance inor near the liver's stem cell niche, aggregate to form spheroid-likestructures on some of the matrix components (e.g., laminins) and spreadinto monolayers on others (e.g., type III collagen). Specific types ofextracellular matrix components, found in the stem cell niche, are amongthe signals requisite for hepatic progenitor cells to undergo expansionin self-replication mode, that is symmetric cell divisions (the daughtercells are identical or nearly identical to the parent cells).

It is further believed that the maturation of hepatic stem cells occursconcomitantly with a unique combination of matrix components whichdirect, at least in part, their differentiation. Some extracellularmatrix components are permissive for hepatic progenitors to undergoexpansion associated with asymmetric divisions, that is expansion alongwith some differentiation. Yet others, located in regions of the livertissue in which fully mature liver cells are found, elicit growth arrestand full differentiation of the cells.

All the feeders produce multiple categories of matrix components andthat include basal adhesion molecules (fibronectin and/or laminin) andseveral collagens. Fibronectin proved to be a matrix component that wasnot expressed by the angioblasts or quiescent hHpSTCs but was expressedby all other feeders studied. It was produced at the highest levels byhuman umbilical vein endothelial cells (hUVECs) but the HpSCs do notattach well to it.

So, its presence in the matrices appears to be irrelevant to thebiological responses induced by the feeders. The feeders that inducedself-replication expressed type III and IV collagens, laminin andhyaluronans (angioblasts, quiescent HpSTCs, HUVEC cells). The feedersthat induced lineage restriction to hepatoblasts and with continuedexpansion produced type IV collagen and laminin but not type III, somehyaluronans, and some chondroitin sulfate proteoglycan (primary culturesreplete with activated HpSTCs, identified by elevated levels of αSMA andCD146). The feeders that induced the maximum differentiation expressedthe highest amounts of matrix and that included high levels of type Iand IV, laminin, fibronectin, and heparan sulfate proteoglycans.

Chondroitin sulfate proteoglycan (CS-PG) protein was evident in bothhuman fibroblast-like, fetal liver-derived cells and bone marrow-derivedmesenchymal stem cells (hMSCs). These two types of feeders causedlineage restriction of hHpSCs into hepatoblasts. Hence, CS-PGs likelysignal, at least in part, that process. It has been hypothesized thatthe stem cell niche is dominated by glycosaminoglycans (GAGs) withlittle to no sulfation such as hyaluronans and these minimally sulphatedCS-PGs could, therefore, act as a barrier minimizing the presentation ofsignals to the stem cells. As the stem cells are pushed out of theniche, they come into contact with GAGs and proteoglycans with moreextensive sulfation and bind growth factors that could influence thestem cells either with respect to growth or with respect to lineagerestriction to various differentiated cell fates.

The most extensive differentiation was observed in hHpSCs plated ontoSTO feeder cells, upon which the hHpSCs went into growth arrest anddifferentiated into hepatoblasts and unipotent progenitors (i.e.,committed biliary and hepatocytic progenitors). The STO feeders producedthe highest levels of extracellular matrix proteins and were unique inproducing HS-PGs.

Type I collagen was determined to induce the most extensivedifferentiation. The extent of differentiation was found to differdepending on whether the cells were plated on top of or embedded intothe type I collagen gel. Indeed, cells morphologically similar to maturehepatocytes were found in those cultures embedded in the collagen (FIG.10). This phenomenon is likely due both to a direct effect of type Icollagen and also an indirect effect via stabilization of HS-PGs by typeI collagen.

FIG. 11 summarizes the discovered sequential changes in matrixcomponents and matrix receptors that occur with the transition of hHpSCsthrough hepatoblasts and ultimately to mature parenchymal cells. Theinvention thus defined can allow the propagation of HpSCs in“feeder-free” cultures. Table 2 provides details on the extracellularmatrix components produced by different feeders studied.

TABLE 2 Extracellular Matrix Components Produced by Different FeedersHuman Fetal Liver Cells Immunoselected for: hHpSCs Depletion of MatrixComponents on plastic hUVECs hMSCs CD31+ KDR+ stroma STO Cells Type INeg Neg +/+ +++ Collagens Type III ++ Neg ++ Type IV ++ + − ++ +++Adhesion Laminin + − − + + + ++ Molecules Fibronectin Neg Pos +++Proteo- Syndecan Neg Neg ++ glycans (HS-PG) Perlecan Neg Neg ++ CS-PGNeg − + − − + +++ GAGs Hyaluronans ++ Functional Effects of FeedersSelf-replication; Lineage restriction to hepatoblasts and with Growth onhHpSCs minimal, if any, considerable expansion arrest; differentiationdifferentiation GAGs = glycosaminoglycans. The antibodies used were: aset from Sigma (Sigma, St. Louis, MO): anti-human type I collagen mouseIgG1, anti-human type III collagen mouse IgG1, anti-human laminin mouseIgG1, anti-chondroitin sulfate mouse IgM. anti-human fibronectin mouseIgG1 (Oncogene Research Products, Cambridge, MA), rabbit anti-human typeIV collagen IgG (Research Diagnostics Inc., Flanders, NJ), ratanti-human perlecan IgG2a (Lab Vision, Fremont, CA), anti-human syndecanControls: antibodies were screened against purified matrix components

The scope of the present invention should not be limited to any onematrix component, soluble component, or combination thereof. In keepingwith the teachings herein, the present invention describes and teachesthe use of any and all soluble and insoluble components and theircombination in the generation of substrata and media that can beutilized for ex vivo maintenance of cells either for expansion or fordifferentiation. While many of these components will be discussed below,for the sake of clarity, laminins, type IV collagens and/or type IIIcollagens will be discussed as mere representatives of a class ofextracellular matrix components that are found in or in high abundancein embryonic tissues or in stem cell niches.

Non-limiting examples of embryonic matrix components include: specifictypes of collagens, including Collagens Type IV (further including α1,α2, α3, α4, α5, α6) and Collagens Type III; Laminins (including, 1, γ1,β2, β3, α5); hyaluronans; forms of chondroitin sulfate proteoglycans(PGs) or their glycosaminoglycan chains; and forms of heparansulfate-PGs or their glycosaminoglycan chains (e.g., certain syndecans).Non-limiting examples of matrix components found in mature tissuesinclude stable forms of collagens (e.g., type I and II), forms offibronectin; heparan sulfate-PGs (e.g., agrin, perlecan), heparin-PGs;dermatan-PGs (e.g., cartilage-associated dermatan sulfate-PG); andelastins.

In addition to insoluble factors, soluble growth and/or differentiationfactors can influence the rate of cell proliferation and/ordifferentiation. For example, the addition of serum can slow growth ofthe hepatic progenitors and cause lineage restriction towards thehepatocytic fate and, in parallel, cause rapid expansion of mesenchymalcell populations (stroma and endothelia) associated with scar tissueformation. The addition of epidermal growth factor leads to lineagerestriction towards an hepatocytic fate.

Preferably, in some embodiments, the matrix components described hereinare employed in combination with a serum-free medium. A serum-free mediawas developed previously for HpSCs and hepatoblasts and is described inU.S. patent application Ser. No. 09/678,953, the disclosure of which isincorporated herein in its entirety.

The present inventors have found that Interleukin-11 (IL-11) andleukemia inhibitory factor (LIF) promoted colony formation of rathepatic progenitor (rter6) cells on top of STO feeders. Because bothIL-11 and LIF are members of IL-6 cytokine superfamily, these findingssupport the notion that the IL-6 cytokine family promotes growth ofhepatic progenitor cells in vitro. EGF reduced colony formation of rathepatoblasts but increased colony formation of diploid adult rathepatocytes but with lineage restriction towards hepatocytes andinhibition of biliary epithelia. As well, TGF-β1 increased colony numberand area of rter6 cells when grown atop STO feeders, but inhibitedgrowth of HepG2 cells on plastic.

Co-culture of hHpSCs and STO feeder cells induced higher expression ofseveral human and mouse cytokines including in majority the inflammatorysignals and some factors known to be hepatic growth stimulating.Interleukin-4 (IL-4) is one of the inflammatory cytokines that elevateddramatically in the co-culture. These and other soluble factors arediscussed with greater detail herein.

Without being held to or bound by theory, it is presently believed thatthe matrix components and soluble components of the present inventionprovide many of the survival, proliferation and/or differentiationsignals generally provided by feeder cells. Thus, the instant inventionmay replace, in significant part, the need for embryonic stromal feedercells to maintain viability and expansion potential of the hepaticprogenitors.

Embodiments of the instant invention will now be described by way ofnon-limiting examples.

EXAMPLES Kubota's Medium (KM)

All cultures were put into KM, a serum-free medium (unless otherwisenoted) tailored for hepatic progenitors. The media is described in, forexample, U.S. patent application having Ser. No. 09/679,663 filed Oct.3, 2000, the disclosure of which is incorporated herein in its entiretyby reference.

Sourcing of Cell Lines

hMSCs were obtained from a 26-year-old male donor. hUVECs were obtainedfrom Dr. Cam Patterson (University of North Carolina; Chapel Hill,N.C.). A clone of murine embryonic stromal cells (STO cells) wasprepared from STO cells obtained from the ATCC.

Sourcing of Human Liver Tissue

Human fetal livers, 16-20 weeks gestational age, were obtained fromAdvanced Biological Resources (ABR, San Francisco, Calif.).

Isolation and Culture of hHpSCs

Human fetal livers were processed as noted, supra. Freshly isolatedparenchymal cells were placed into KM and culture plastic or atoppre-plated feeders of hUVECs, hMSCs, STO cells, or primary cultures ofhuman fetal liver-derived cells at a plating density of 5,000 cells/cm².The cells were in KM plus 2% FBS overnight and then switched to KMthereafter. The cultures on plastic and in KM yield colonies of hHpSCssurrounded by angioblasts and hHpSTC precursors that were not activated.

Preparation of Feeders

All stocks of mesenchymal feeders were cultured on culture plastic andin Endothelial Growth Medium, (EGM-2) (Cambrex, Walkersville, Md.) with2% FBS. The only exceptions to these conditions were the hMSCs and theadult liver-derived HpSTCs, which were grown as described below. Allcells were grown to confluence, growth arrested with Mitomycin-C, andthen switched to KM for use in co-cultures with hHpSCs. Further detailsfollow:

hMSCs were plated onto tissue culture dishes with DMEM plus 1%antibiotics, ascorbic acid, 2 mM L-glutamine and 10% FBS.

Purified preparations of HpSTCs from adult rat and adult human liverswere prepared by Dr. YiWei Rong. The stocks of feeders were cultured onplastic and in KM+5% FBS.

STO5 feeders were cloned from STO cells obtained from the ATCC and weretested for their efficacy on rodent hepatic progenitors. Frozen stocksof STO5 were thawed and grown in KM to which 5% fetal bovine serum wasadded.

For preparation of primary cultures of human fetal liver-derivedmesenchymal cells, livers were enzymatically digested using 0.45 mg/mlcollagenase type IV and 0.3 mg/ml deoxynuclease and then mechanicallydissociated into single cell suspensions by cross scalpels. Afterwashing away excess enzymes, the cells were put through three rounds ofslow-speed centrifugation (20×g) for 5 minutes. The supernatant wascollected and resuspended in RPMI-1640 plus selenium (10⁻⁹M), 1%antibiotics and 0.1% BSA. The cells were then plated onto cultureplastic and in KM supplemented with 10% FBS. The mesenchymal cellsattached within minutes to hours and quickly transitioned into stromalfeeders comprised of activated hHpSTCs recognizable by having highlevels of desmin, CD146 and αSMA.

KDR+ or CD31+ cells were isolated from the fetal liver cell suspensionsby magnetically activated cell sorting (MACS) system using monoclonalanti-human KDR mouse IgG1 (Cell Sciences, Canton, Mass.), goatanti-mouse IgG coupled to magnetic microbeads or using monoclonalanti-human CD31 mouse IgG1 conjugated to magnetic microbeads. Platingdensity for KDR⁺ and CD31⁺ cells was 20,000 cells/cm².

Feeders depleted of stromal cells were prepared by negative selectionfor fibroblasts using monoclonal anti-human fibroblast mouse IgG2aconjugated to magnetic microbeads according to manufacturer'sinstructions (Miltenyi Biotec, Auburn, Calif.). The plating density forfibroblast-depleted supernatant cells was 500,000 cells/cm².

Purified Matrix Substrata

The preparation of matrix substrata for in vitro culture is described inU.S. patent application having Ser. No. 11/560,049 filed Nov. 15, 2006,the disclosure of which is incorporated herein in its entirety byreference.

Fibronectin: Fibronectin (Sigma, F0895) was coated onto dishes atconcentrations of 0.5, 1.0, or 2 μg/cm² and then neutralized to pH 7.4.

Laminin: Laminin (Sigma, L2020) was coated onto dishes at concentrationsof 0.52 or 1.0 μg/cm² at pH 7.4.

Collagen, types III and IV: Collagen coatings were prepared on dishes at1 of 5 different protein concentrations (2.1, 4.2, 6.3, 8.3, and 10.4μg/cm²). Matrix components were added in acidic buffers to the dishes.The matrix was allowed to attach over a 10-hour period at 37° C. and 5%CO₂. After 10 hours, the dishes were sterilized by UV irradiation for2-hours and then rinsed 3× with PBS. Collagen III (Sigma, C-3511) wasformed with pH 3 acetic acid and Collagen IV (Sigma, C-5533) with 0.5Macetic acid.

Collagen, type I: Vitrogen 100 (Cohesion Technologies, Palo Alto,Calif.) was modified into liquid collagen type-I by adding specificratios of 10×DMEM and 0.1M NaOH. Because air bubbles can make gelsunstable, the formation of air bubble formations was prevented. Thecollagen I was used both for monolayers of cells or as a “sandwich” toembed cells between two layers of collagen.

Monolayers of Cells on Collagen I: Liquid collagen I was maintained at4° C. prior to distributing 0.4 ml into each well of a 6-well plate.After coating, the collagen was gelled at 37° C. and 5% CO₂ for 1-hour.

Sandwich (Embedded cells) Model: Cells were sandwiched between layers ofcollagen. After a 10-hour period for cell attachment period, unattachedcells were removed, and a second 0.4 ml layer of collagen I added. Thesystem was allowed to gel at 37° C. and 5% CO₂ for 1 hour to solidifythe top collagen layer.

Immunohistochemistry on Human Hepatic Progenitor Cells and Human FetalLiver-Derived Feeder Cells

After 1-2 weeks of culture, cells were fixed with 4% paraformaldehydefor immunostaining. A antibodies used were as follows: FITC-conjugatedanti-human vWF sheep IgG (US Biologicals, Swampscott, Mass.),PE-conjugated anti-human CD56 (NCAM) mouse IgG1, anti-human CD31 mouseIgG1, PE-conjugated anti-human CD54 (ICAM-1) mouse IgG1 (BD, San Jose,Calif.), anti-human αSMA mouse IgG2a, anti-human type I collagen mouseIgG1, anti-human type III collagen mouse IgG1, anti-human laminin mouseIgG1, anti-chondroitin sulfate proteoglycan mouse IgM (Sigma, St. Louis,Mo.), anti-human fibronectin mouse IgG1 (Oncogene Research Products,Cambridge, Mass.), rabbit anti-human type IV collagen IgG (ResearchDiagnostics Inc., Flanders, N.J.), rat anti-human perlecan IgG2a (LabVision, Fremont, Calif.), rabbit anti-human AFP IgG (Zymed-Invitrogen,South San Francisco, Calif.), anti-human KDR mouse IgG1 (Cell Sciences,Canton, Mass.), Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 568goat anti-rabbit IgG, Alexa Fluor 568 goat anti-mouse IgG1 and AlexaFluor 488 goat anti-mouse IgG2a (Molecular Probes-Invitrogen, Eugene,Oreg.).

Quantitative Real-Time PCR

Total RNA was extracted from cells using RNeasy® Mini (Qiagen, Valencia,Calif.). The extracted RNA was then reverse-transcribed into cDNA usingSuperScript® II RT (Invitrogen, Carlsbad, Calif.). Real-timequantitative PCR was performed using sequence specific primers andprobes shown in the Table 3 below and analyzed by the ABI Prism 7000Sequence Detection System (Applied Biosystems, Foster City, Calif.,USA). Ribosomal RNA (18S) from each cell type was used as internalcontrol. The mRNA expression levels relative to 18S were determined andthe fold changes were calculated using the 2-^(ΔΔCT) method. Primersused are tabled below:

TABLE 3 PCR primers Genes ABI assay number Ribosomal RNA (18S) 4308329type I collagen-α1 chain (Col1A1) Hs00164004_m1 type III collagen-α1chain (COL3A1) Hs00164103_m1 type IV collagen-α1 chain (Col4A1)Hs00266237_m1 type V collagen-α1 chain (COL5A1) Hs006090088_m1fibronectin module 1 (FN1) Hs01549972_m1 laminin-α2 chain (LAMA2)Hs00166308_m1 laminin-α4 chain (LAMA4) Hs00158588_m1 laminin-α5 chain(LAMA5) Hs00245699_m1 laminin-β1 chain (LAMB1) Hs00158620_m1 laminin-γ1chain (LAMC1) Hs00267056_m1 syndecan-1 (SDC1) Hs00174579_m1 syndecan-2(SDC2) Hs00299807_m1 glypican-3 (GPC3) Hs00170471_m1 glypican-5 (GPC5)Hs00270114_m1“Native” Feeder Cells for hHpSCs

Freshly isolated hHpSCs survived ex vivo on tissue culture plastic inKM, when the cells were in the presence of angioblasts (VEGFR2+, CD31+,CD133/1+, CD117+) and quiescent hHpSTCs (CD146-low, desmin and αSMA)(FIGS. 1-3). Colonies of hHpSCs consisted of cells tightly bound to eachother on their lateral borders but with minimal attachment to theculture dish per se. However, at the perimeter of the colonies, the siteat which the angioblasts cells were located, the hHpSCs attached to thedish. Thus, attachment was either by the mesenchymal companion cells(e.g., the angioblasts) alone or in combination with hHpSCs.

Feeder Cell Lines and Feeder Primary Cells Used to Model the “Native”Feeders

Several forms of embryonic mesenchymal cells, either primary cultures orcell lines, were prepared as models of “native” feeders (e.g.,angioblasts and HpSTCs). hHpSCs were cultured on these feeder cells inKM. While minimizing exposure to serum is essential to stave offspontaneous differentiation of hHpSCs, mesenchymal feeders requirefactors from serum for survival. To overcome this technical hurdle, thepresent inventors grew stocks of mesenchymal feeders in medium, such asEGM-2, supplemented with 2% serum before switching to serum-free medium,such KM, for assays requiring co-culture of feeders and hHpSCs.

When maintained in serum-free medium (FIGS. 1-3), hHpSTCs were found tobe quiescent, expressing low levels of CD146, desmin and αSMA. Uponexposure to serum, however, even at low (1-2%) levels or for 5 days,resulted in activation of the hHpSTCs as evidenced by high levels ofCD146, desmin and αSMA (FIG. 4). Exposure to serum also induced primarycultures of fetal liver cells, or immunoselected cells (i.e., KDR+ orCD31+ cells, discussed hereinbelow), to differentiate into hHpSTCs.

Feeder cell lines tested were: hMSCs (FIG. 5A), hUVECs (FIG. 5B), andmurine embryonic stromal cells (STO), often used for maintenance of EScells in culture. Primary cultures were prepared from cell suspensionsof 16-20 week-old human fetal livers by immunoselection. Cellsexpressing KDR (a.k.a. flk-1/VEGFR2) or CD31 (a.k.a. PECAM), or cellsthat remain upon negative sorting for fibroblasts thereby reducing oreliminating stromal cells were selected by MACS. Whole livers comprised0.5% and 1% KDR+ and CD31+ cells, respectively. These immunoselectedpopulations of cells were cultured in EGM-2 medium.

Immunoselected KDR+ cells changed rapidly in culture. In the first week,the cells morphologically and antigenically appeared as angioblasts orendothelial cells (FIG. 3D). By the second week, however, HpSTCsdominated the culture. Indeed, the cultures were confluent at 11 days,and most of the cells were positive for αSMA, a marker for hHpSTCs andnegative for vWF, an intracellular marker for endothelial cells (FIGS.5F and H and 6I and K). Even if the cell suspension was negativelyfractionated to eliminate stroma prior to plating, the same phenomenonwas observed (FIGS. 5C-E and G).

CD31+ cells appeared as cobblestone-like cells in morphology for thefirst five days in culture and were positive vWF, indicating that thecells were endothelial cells. After 5-7 days of culture, however,hepatic stellate cells (strongly expressing αSMA and desmin) dominatedthe dish and quickly reached confluency by day 9-10. The resultsdemonstrate that EGM-2, though specifically designed for endothelialcells is nevertheless permissive for outgrowth of hHpSTCs.

hHpSCs on Feeders of Angioblasts or hUVECs Remain as Stem Cells

Isolation and clonogenic expansion of hHpSCs on culture plastic and inKM in which there was close association with angioblasts and quiescentHpSTCs resulted in cells that remained as hHpSCs with minimaldifferentiation (FIG. 3). The hHpSC colonies can be seen 2 weeks afterplating and are positive for NCAM, EpCAM, albumin, CK19 and CLDN-3 andnegative for AFP. hHpSCs cultured in KM and atop hUVECs or on KDR+feeder cells immediately after sorting also maintained hHpSCs as stemcells (FIG. 7B) with an antigenic profile of EpCAM+, NCAM+, ICAM-1−,AFP−, CLDN-3+.

hHpSCs Cultured on Feeders of Activated Hepatic Stellate Cells LineageRestrict to Hepatoblasts

hHpSCs cultured on activated hHpSTCs caused rapid transition, withinhours, of hHpSCs to hepatoblasts (FIG. 4). hHpSCs cultured on eitherhMSCs; primary human fetal liver stroma cells; primary human fetal liverstroma cells depleted of fibroblasts; primary KDR+ cells; or primaryCD31+ cells also transitioned to hepatoblasts after more than a week inculture. After 8-9 days of co-culture with any of these feeders, thehepatic progenitor colony morphology consisted of cord-like structuresinterspersed with clear channels, the presumptive biliary canaliculi(FIGS. 7 and 8) and with an antigenic profile indicative of hepatoblasts(EpCAM+, NCAM−, ICAM-1+, AFP+) (FIG. 1). Moreover, the morphology ofhepatoblast colonies was more 3-dimensional causing them to berefractile when evaluated by bright field (FIGS. 7 and 8) possiblycaused by multiple layers of cells and/or accumulation of extracellularmatrix.

hHpSCs Plated onto STO Feeders

The feeder model system resulting in the maximum differentiation provedto be STO feeders. hHpSCs plated onto these feeders significantly slowedtheir growth and then gave rise to hepatoblasts and committedprogenitors from the edges of the colonies.

Gene Expression of Matrix Molecules by the Feeders

Three feeder cell types were chosen to represent feeders that eithersustained the hHpSC phenotype (hUVEC cells); caused differentiation tohepatoblasts (primary cultures of human fetal liver mesenchymal cellsand CD31+ cells); or lead to more advanced differentiation down thehapatocytic pathway (fetal liver-derived endothelia cultured for morethan a week, both assayed at time points at which hHpSTCs were thedominant cell population). Using real time PCR, it was found thatfibronectin mRNA encoding the type I module of the fibronectin moleculewas the highest-expressed matrix component among the three feedersassayed, especially in hUVECs (FIG. 9). The hUVEC feeders, supportive ofmaintenance of the hHpSC phenotype produced collagen type IV, laminin(α4, β1 and γ1 chains), and little or no collagen types I and III,laminin chain isoforms other than those aforementioned, or proteoglycancore proteins. Those that induced lineage restriction to hepatoblasts(human fetal liver-derived αSMA+fibroblast-like and CD31+ cells)produced type I, III and IV collagen, laminin (β1, γ1), but not collagentype V, other laminin chains or any of the proteoglycan core proteingenes assayed (Glypican-3 and -5 and Syndecan-1 and -2).

Protein Expression of Matrix Molecules by the Feeders

Immunohistochemistry (IHC) was performed on the feeders for 7 differentmatrix molecules: type I, III and IV collagens, laminin, fibronectin,and heparan sulfate proteoglycans (HS-PG) (i.e., perlecan and syndecan)and chondroitin sulfate proteoglycans (CS-PG). All feeders produced amix of extracellular matrix molecules, but the lowest levels of totalmatrix molecule production were found in primary cultures ofangioblast/endothelia; followed by the hUVEC cell line or primarycultures in which hHpSTCs had been culture selected. The highest levelof matrix molecules were produced by STO cells. The basal adhesionmolecule, fibronectin, was found in all of the feeders and with thehighest levels found in STO cells. Interestingly, collagen type III wasfound expressed only in STO cells, whereas it was found in other feedersby RT-PCR.

Without being held to nor bound by theory, hHpSCs maintain theirphenotype when cultured on an extracellular matrix containing forms oflaminin with integrins α4 and β6, type IV collagen, CS-PG, and noHS-PGs. Matrices that induce lineage restriction to hepatoblasts haveelevated levels of type I, III and IV collagens, laminin (no α4 and noincrease in the β1 isoform), CS-PG and no HS-PGs. Finally, matrices thatinduce the most striking differentiation (i.e., beyond the hepatoblastsstage) also have all of the matrix components mentioned but levelshigher than observed among other feeders. These matrices, however, wereunique in containing HS-PGS (FIG. 11).

Effects of Purified Matrix Molecules on hHpSCs Versus Hepatoblasts

hHpSCs were cultured in KM and on each of the following 5 types ofmatrix components coated onto plastic dishes: fibronectin, laminin andtype I, III or IV collagen. Few of the hHpSCs cells attached tofibronectin, and those that attached did not grow. hHpSCs lineagerestricted to hepatoblasts if cultured on laminin and/or type IVcollagen or if plated on a surface of type I collagen gel (FIG. 10).When embedded into type I collagen, the hHpSCs differentiated the most,with the morphology and antigenic profile of the colonies resemblingthat of mature hepatocytes.

Matrix components within the periportal zone and in the liver's stemcell niche are distinct from those found in association with the matureparenchymal cells and elicit distinct biological responses from purifiedsubpopulations of human hepatic stem/progenitor cells. These differencesare likely to provide diverse signals that modify cell responses andactivate dynamic expressions. By determining how distinct classes ofextracellular matrix components induce in vivo and in vitro cellactivities, microenvironments can be reproduced in vitro to expand anddifferentiate HpSC populations for the replacement or repopulation ofdiseased tissues.

In addition to matrix proteins, feeder cells are thought to providesoluble factors (e.g., cytokines, growth factors) that are essential forHpSC survival, proliferation, and/or differentiation. Non-limitingexamples of relevant factors are listed below:

Eotaxin (human and Eotaxin: encoded by the CCL11 gene in human (Ccl11 inmouse), known to attract eosinophils in the mouse) presence ofallergens. G-CSF (mouse) Granulocyte Colony Stimulating Factor. Encodedby the mouse gene csf3, expression this 208 aa protein is known tostimulates the growth and differentiation of hematopoietic precursorcells from granulocytes, macrophages, eosinophils and erythrocyteslineages. GM-CSF (human Granulocyte-Macrophage Colony-Stimulating.Encoded by the CSF2 gene in human, (csf2 in and mouse) mouse) thisprotein is indispensable for the growth and development of progenitorsof granulocytes and macrophages, and triggers the irreversibledifferentiation of myeloblasts and monoblasts. HGF (human) HepatocyteGrowth Factor. Encoded by the human gene HGF (give rise to the 728 aaprecursor) HGF is a growth factor for a broad spectrum of tissue andcells. HGF is involved in the maturation of liver stem cells. IFN-γ(human and Interferon gamma. Encoded by the human gene IFNG (mouseIfng), IFN-γ is a dimeric protein mouse) which comprises six naturallyoccurring variants forms. In addition to its immunomodulatoryactivities, IFN-γ is a growth-promoting factor for T-lymphocytes and agrowth inhibitor for IL-4 induced B-cells, smooth muscle cells andendothelial cells. It functions as an inhibitor of capillary growthmediated by myofibroblasts, fibroblast growth factors and PDGF. KC(Mouse) Keratinocyte Chemoattractant. The new designation for KC isCXCL1 and the gene symbol is Cxcl1. KC is involved in chemotaxis andcell activation of neutrophils. In vitro it inhibits hematopoieticprogenitor cell proliferation. A natural N-terminally truncated form (4amino acids) can be isolated from bone marrow stromal cells. This factoraugments the formation of Granulocytes- Macrophage Colonies FormingUnits in the presence of other colony stimulating factors. Compared withfull-length KC, the shortened factor is approximately 10 million timesmore potent as synergistic growth stimulant for GM.CFU. IL-1RaInterleukin 1 receptor antagonist. Encoded by the IL1RA gene The 152 aasecreted protein and the (human) 177 intracellular forms antagonize IL-1by binding to the IL-1 receptor. IL-1α (human) Interleukin 1alpha.Encoded by the gene IL1A, the 271aa precursor is processed by a highlyregulated mechanism to a159 aa secreted protein. IL1 in combination withother cytokines is an important mediator of inflammatory reactions. infibroblasts, synovial cells, chondrocytes, endothelial cells,hepatocytes, and osteoclasts. IL1 acts directly on B-cells, promotingtheir proliferation as well as that of fibroblasts, thymocytes, andglioblastoma cells. IL1 inhibits the growth of endothelial cells invivo. In vascular smooth muscle cells and skin fibroblasts IL1 inducesthe synthesis of bFGF which is a mitogen for these cells. IL-1β (humanand Interleukin 1 beta. Encoded by the human gene IL1B (mouse geneIl1b). The Activities or IL1-beta mouse) are similar to IL1alpha. IL-2(human and Interleukin 2. This protein, encoded by human gene IL2 andmouse gene Il2 induces clonal mouse) expansion of Tcells. IL-4 (humanand Interleukin 4 encoded by Human gene IL4 and mouse gene Il4, it hasbeen shown to promote the mouse) proliferation and differentiation ofactivated B cells, and synergize with Epo and G-CSF in generatinggranulocytes and erythroid progenitor cells colonies. A naturallyoccurring splice variant designated IL4-delta2 act as an antagonist.IL-5 (human and Interleukin 5. Encoded by human gene IL5 and mouse geneIl5, this protein is a specific mouse) hematopoietic factor thatstimulates the growth and differentiation of eosinophils, as well as theproliferation of immature hematopoietic progenitor cells andproliferation and differentiation of immature B cells. IL-6 (human andInterleukin 6 encoded by human gene IL6, mouse gene il6. This proteinstimulates specific and mouse) innate immune responses. It activates theexpression of acute phase proteins in hepatocytes. IL6 and IL3 synergiseto promote the proliferation of multipotent hematopoietic progenitorcells. IL6 is a B- cell differentiation factor in vivo and an activationfactor for T-cells. IL6 is expressed early in murine blastocystssuggesting that it may regulate the growth and development oftrophoblasts or embryonic stem cells. IL-8 (human) Interleukin 8.Encoded by the IL8 gene, this protein is found under several variantsforms (from 69 aa to 77 aa). In addition its chemotactic activities IL8is a mitogen for epidermal cells. Macrophage- derived IL8 supportsangiogenesis in physiological situation such as wound repair. IL-10(human and Encoded by the human IL10 gene (Il10 mousegene) this cytokineis a chemoattractant of CD8 mouse) cytotoxic cells and downregulator ofthe expression of several cytokines. IL-11 (human and Interleukin 11,encoded by human gene IL11, (mouse gene Il11) is a cytokine which inaddition to mouse) promoting immune responses, stimulates severalmultilineages hematopoietic progenitors, inhibits the differentiation ofadipocytes progenitors and induces the synthesis of acute phase proteinsin hepatocytes. IL-12 (human and Interleukin 12. A heterodimeric proteinencoded by the human genes IL12A and IL12B (mouse mouse) genes Il12a andIl12b), it is known to stimulate the proliferation of lymphocytes andsynergizes with several factors to promote myelopoiesis of bone marrowprogenitor cells. IL-13 (human) Interleukin 13. This 132 AA proteinencoded by IL13 attenuates the inflammatory response. It also is knownto induce differentiation of monocytes, and modulate proliferation,differentiation and Isotype switching of Bcells. LIX (mouse)Lipopolysaccharide-Induced CXC chemokine, 92 aa equivalent of humanGCP2/ENA78, Renamed CLCX5, encoded by the clcx5 gene. Known as amonocytes chemoattractant produced in response to bacterial LPS. MCP-1(human and Monocyte Chemotactic Protein 1. Renamed CCL2 and encoded bythe human gene CCL2 and the mouse) mouse Ccl2 gene, this protein ischemotactic for monocytes but not neutrophils. In addition tochemotaxis. MCP-1 can induce the proliferation and activation of killercells known as CHAK (CC- Chemokine-activated killer). MCP-2 (human)Monocyte chemotactic protein 2. Renamed CCL8, encoded by human geneCCL8, this chemotactic factor attracts monocytes, lymphocytes, basophilsand eosinophils. MIP-1α (mouse) Macrophage Inflammatory Protein 1a.Encoded by Ccl3, This 92 aa protein induces synthesis of IL- 1 IL-6 andTNF by fibroblasts, Synergizes with GM-CSF in promoting maturation ofhematopoietic progenitor cells, while inhibiting the proliferation ofhematopoietic stem cells. MIP-1β (human) Macrophage Inflammatory Protein1beta. Encoded by CCL4, this 92 aa protein induces inflammatory responseand promotes the maturation of hematopoietic progenitor cells. MIP-2(mouse) Macrophage Inflammatory Protein 2. Encoded by the Cxcl2 gene,this 73 aa protein is chemotactic for polymorphonuclear lymphocytes.RANTES Regulated upon Activation Normal T-cell Expressed Secreted alsocalled CCL5, this chemokine is chemotactic for T-cells, eosinophils andbasophils. It also activates and proliferation of Killer cells. sTNFR2Secreted Tumor necrosis factor receptor 2. The gene TNFRSF1B encodes themembrane bound 439 aa protein which is cleaved to a believed 235aa. Thesoluble form of the receptor antagonizes activity of TNFalpha by itsability to store and sequester it. sTNFR1 Secreted tumor necrosis factorreceptor 1 encoded by the gene TNFR1A, the 251 aa soluble form of theTNF-α receptor 1 is thought to antagonize action of TNF- α bysequestering it. TNF-α (human and Tumor Necrosis Factor alpha is encodedby the human gene TNFA and mouse gene Tnfa TNF-a mouse) shows a widespectrum of biological activities. In addition to its participation ininflammatory processes, TNF is a potent promoter of angiogenesis in vivowhich is antagonized by IFN-gamma. It is also a growth factor for normalhuman diploid fibroblasts, astroglial cells and microglial cells.Effects of Mesenchymal Cells-Conditioned Media on Rat Hepatic Progenitor(rter6) Cell and Human Hepatoblastoma (HepG2) Cell Colony Formation

rter6 are unable to generate colonies efficiently on inert substratasuch as plastic or on extracellular matrix-coated plates but can producecolonies when plated on STO feeders. Hence, experiments were conductedto determine how well rter6 cells could grown in media “conditioned” bySTO feeders. To generate “conditioned” media, stocks of feeders (STOcells or a human fetal lung fibroblast cell line (MRC5)) were grown inserum-supplemented medium until confluence, rinsed to remove any serum,and then switched to KM, which is serum-free. The cells were allowed togrow for another 48 hours in the serum-free medium, thus “conditioning”it with factors produced by the feeders. This conditioned media was thenused in the experiments.

The colony numbers of rter6 cells co-cultured for 10 days with STOfeeder cells and STO conditioned medium increased 2.39-fold comparedwith KM (Tables 4 and 5). The number of rter6 colonies when co-culturedfor 10 days with MRC5 feeder cells and STO conditioned medium increased1.57-fold compared with KM (Tables 4 and 5). In serum-free HDM, MRC5cells appeared to promote more colony formation of rter6 cells comparedwith STO feeders. In STO conditioned medium, rter6 cells on both feedershad the similar colony formation ability.

TABLE 4 Effect of STO conditioned-medium on colony formation of rter6cells cultured on STO or MRC5 feeder Rter6 seeding 100 200 400 Averagefold density (cells/cm²) increase of Average HDM 7.25 15.25 27.25 colonyno.: colony no. +STO-CM 18 34.5 66 2.39 Fold increase 2.49 2.26 2.42P-value 0.0065 0.0009 0.0003

TABLE 5 Effect of STO conditioned-medium on colony formation of rter6cells cultured on MRC5 feeder Rter6 seeding 100 200 400 Average folddensity (cells/cm²) increase of Average HDM 14.75 23.5 42.5 colony no.colony no. +STO-CM 23 37.25 66.5 1.57 Fold increase 1.56 1.59 1.56P-value

Human hepatoblastoma (HepG2) cells can form colonies on uncoated tissueculture plastic. Serum-free conditioned media from four different feedercell types were used to test the colony formation of HepG2 cells: (1)STO cells; (2) MRC5 cells; (3) immortalized adult human hepatic stellatecells (h-tert-HpSC); and (4) primary human fetal liver-derived stromalcells. Compared with HDM, the serum-free STO-conditioned mediumincreased colony formation of HepG2 cells, while the serum-free mediaconditioned by MRC5 cells, h-tert-HpSC or primary human fetalliver-derived stromal cells inhibited HepG2 cell colony formation.

ELISA on Conditioned Media from STO Cells, Human Fetal Liver Cells, andCo-Cultures of Both

The concentration of 23 human cytokines, 17 mouse cytokines and 2non-species specific cytokines were tested on cultured media conditionedby STO feeder, human fetal liver-derived progenitor cells and co-cultureof both. For human cytokines, increased concentration of solubleinterleukin-1 receptors (IL-1Rα), interleukin-1α (IL-1α), IL-2, IL-4,IL-5, IL-10, IL-12, IL-13, macrophage chemoattractant protein-2 (MCP-2),eotaxin, soluble tumor necrosis factor receptor-2 (sTNF-R2), andregulated on activation, normal T-cell expressed, and presumablysecreted (RANTES) was observed in co-culture compare with human fetalliver-derived progenitor cells cultured alone (FIG. 12, Table 6).Cytokines with decreased concentration in co-culture were interleukin-11(IL-11), granulocyte macrophage-colony stimulating factor (GM-CSF),macrophage inflammatory protein-1α (MIP-1α), MIP-1β, soluble TNFreceptor 1 (sTNF-R1) and hepatocyte growth factor (HGF) (FIG. 12, Table6).

TABLE 6 ELISA analyses of conditioned medium from STO cells and fromhuman fetal liver cells Concentration (pg/ml) Concentration (pg/ml)STO/hFLC co-culture Mouse STO/hFLCco- Human Human Human and cytokinesSTO culture Cytokines hFLCs cytokine mouse TNF-α 0 1.4 TNF-α 0 0.5 1.9IL-1β 0 3 IFN-γ 0.8 2.5 23.4 IL-12 0 11.3 IL-10 1 4 62.8 IL-2 7.7 12IL-1β 3 4.8 7.8 IFN-γ 0 20.9 IL-5 0 7.5 133.7 GM-CSF 5.8 35.3 IL-12 1 819.3 IL-11 0 46.2 IL-2 0.8 10 22 IL-10 0 58.8 GM-CSF 73 11 46.3 Eotaxin33.96 68.3 IL-13 0 16 n/a G-CSF 2.6 118.7 MCP-2 10 18 n/a IL-5 3.8 126.2IL-1α 13.8 24.1 n/a MIP-2 0 182 IL-4 5.2 34.3 317.8 IL-6 28.2 192.3Eotaxin 15.5 34.4 102.7 MIP-1α 86.7 283.17 IL-1Rα 24 34.7 n/a IL-4 2.3283.5 IL-11 1221 43.1 89.3 LIX 299 499 MIP-1β 292 111 n/a KC 800 794sTNFR2 66 166 n/a MCP-1 2095 2094 IL-8 172 177 971 HGF 399.9 253.8 n/asTNFR1 687 283 n/a TGF-β1 136.3 n/a 287.8 IL-6 327 290 482.3 RANTES 67320 n/a MIP-1α 981 360 643.17 MCP-1 539 531 2625

For mouse cytokines, increased concentration of interleukin-2 (IL-2),IL-4, IL-5, IL-6, IL-10, IL-11, IL-12, granulocyte macrophage-colonystimulating factor (GM-CSF), eotaxin, granulocyte-colony stimulatingfactor (G-CSF), macrophage inflammatory protein-1α (MIP-1α),interferon-γ (IFN-γ) and lipopolysaccharide-induced CXC chemokine (LIX)were observed in co-culture compared with STO cells cultured alone (FIG.13, Table 6). There was no significant decrease of concentration of anymouse cytokines tested in co-culture.

For non-species specific cytokines, transforming growth factor-β1(TGF-β1) was increased in co-culture (532.3 pg/ml) compared with STOcells cultured alone (10.2 pg/ml) and with human fetal liver-derivedprogenitor cells cultured alone (22.3 pg/ml).

Human hepatic stem cells have been found to differentiate intohepatoblasts when co-cultured with STO feeder cells. Combinedconcentration of human and mouse cytokines in co-culture revealed thatIL-4, IL-5, IL-10, CXC chemokine (mouse keratinocyte-derived chemokines(KC) and human IL-8, eotaxin, MCP-1 and RANTES had dramatic increase(≧5-fold and >50 pg/ml) of protein concentration compared with humanfetal liver cells cultured alone (Table 6).

Effects of Soluble Cytokines on rter6 Cells and HepG2 Cells ColonyFormation

Of the cytokines uncovered, nine were added individually intoserum-free, hormonally-defined medium (HDM) and tested. In addition tothe media, the cells were grown on STO feeder layers and incubated for10 days. Leukemia inhibitory factor (LIF, 0.5 ng/ml), interleukin-11(IL-11, 10 ng/ml), and transforming growth factor-β1 (TGF-β1, 0.05ng/ml) increased the colony number and colony area of rter6 cellscompared with controls (FIG. 14). Interleukin-6 (IL-6), interleukin-13(IL-13), hepatocyte growth factor (HGF), growth related oncogene-α(GRO-α), macrophage inflammatory protein-1α (MIP-1α) and tumor necrosisfactor-α (TNF-α) had no observed effect on colony formation of rter6cells.

Several cytokines and candidate stimulatory molecules were also addedindividually into HDM and STO conditioned medium to test their effectson colony formation of HepG2 cells. Hydrocortisone increased colonyformation 25% in both HDM and STO conditioned medium compared withcontrol. Insulin-like growth factor-II (IGF-II), interleukin-6 (IL-6),interleukin-11 (IL-11), interleukin-13 (IL-13), tumor necrosis factor-α(TNF-α), growth related oncogene (GRO; CXC chemokine), human growthhormones, and high density lipoproteins (HDL) had no observed effects onHepG2 cell colony formation. Transforming growth factor-β1 (TGF-β1) hadinhibited HepG2 cell growth and survival. Epidermal growth factor (EGF)significantly decreased HepG2 colony formation and caused the cells tomigrate away from colonies.

Taken together, the present invention enables the survival,proliferation, and/or controlled differentiation of HpSCs in the absenceof feeder cells. The following Table 7 lists non-limiting examples of“feeder-free” conditions for propagating HpSCs in vitro and ex vivo. Allof the examples include hyaluronans, which are ubiquitous in the stemcell niche in livers in vivo. It is thought at present that hyaluronansenhance the efficiency of propagating HpSCs. However, the examplesshould not be construed so as to require the presence of hyaluronans inculturing HpSCs in vitro. See, e.g., U.S. provisional patent applicationno. 60/893,277 filed Mar. 7, 2007, the disclosure of which isincorporated herein in its entirety by reference.

TABLE 7 Hyaluronans with disulfide cross-linking would be complexedwith: Basal Adhesion Proteoglycan Hormones and/or Lineage StageCollagen(s) Protein (or GAG chains) growth factors I. Conditions forExpansion of the Cells Hepatic Stem Cells and/or Type III (and LamininHeparan sulfate Insulin, Hepatoblasts IV) collagen proteoglycan (ortransferrin/fe, LIF, HS) from human FGF4, IL6 (and/or fetal liversIL11), HGF, HDL, free fatty acids on human albumin, and hepatopoietinHepatic Stem Cells and/or Type III (and Laminin Heparan Sulfate Insulin,Hepatoblasts (mixed with type IV) proteoglycan (or transferrin/fe, LIF,angioblasts/endothelia) collagen HS) from human FGF4, IL 6 [grafts]fetal livers (and/or IL11), HGF, VEGF, HDL, free fatty acids bound tohuman albumin, and hepatopoietin II. Conditions for Differentiation ofStem/Progenitor Cells Hepatic stem Type IV Laminin/Fibronectin HeparinInsulin, cells/hepatoblasts collagen (and Proteoglycan (ortransferrin/fe some type I) Heparin) from T3, IGFI, HGF, Adult Humanhydrocortisone, Liver HDL and free fatty acids bound to human albuminIII. Conditions for Growth of Adult Hepatocytes Hepatocytes (diploidcells Type IV Laminin Heparan sulfate Insulin, EGF, T3, must be used forcomplete collagen proteoglycan (or IGF I, HGF, cell division) HS) fromfetal HDL, free fatty human livers acids bound to human albumin,hepatopoietin IV. Conditions for Maximal Differentiation of AdultHepatocytes Hepatocytes (both diploid Type I Fibronectin HeparinInsulin, EGF, T3, and polyploid cells) collagen (with Proteoglycan (orIGF I, HGF, small amounts Heparin) from Hydrocortisone, of type III)Adult human HDL, Free Fatty livers Acids bound to human albumin, andglucagon

In this way, transplanted cells obviate whole organ replacement alltogether. Furthermore, in vitro devices such as bioreactors may beseeded with hepatic progenitors enveloped in an appropriateextracellular matrix and soluble signaling environment so they populatedevice subcompartments with viable tissue structures. In this way,bioartificial devices can be utilized for pharmacology studies, vaccinedevelopments, and as a bridge between organ failure and organtransplantation. Indeed, the results obtained from these investigationssuggest that utilizing these cells may be an avenue to improve cellsourcing limitations that currently inhibit both cell therapy andbioreactor device medical treatments options.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or alterations of the invention following. In general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1. A method of propagating hepatic stem cells in vitro without inducingtheir differentiation comprising: culturing a population of isolatedhepatic stem cells in serum-free culture media and on a layer of matrixcomponents selected from the group consisting of hyaluronans, otherunsulfated or poorly sulfated glycosaminoglycans (GAGs), unsulfated orpoorly sulfated proteoglycans, embryonic collagens and embryonic basaladhesion molecules, and combinations thereof, in which the layer isessentially free of mature collagens, and in which the culturingpropagates the hepatic stem cells without inducing theirdifferentiation.
 2. The method of claim 1 in which any or all of thematrix components are supplied by angioblast feeder cells, quiescenthepatic stellate feeder cells, HUVEC feeder cells, or a combinationthereof.
 3. The method of claim 1 in which the embryonic collagens areof type III.
 4. The method of claim 1 in which the mature collagen iscollagen type I.
 5. The method of claim 1 in which the basal adhesionmolecules comprise isoforms of laminin found predominantly in fetaltissues.
 6. The method of claim 1 in which the GAGs other thanhyaluronans are forms of chondroitin sulfates.
 7. The method of claim 1in which the proteoglycans are forms of chondroitin sulfateproteoglycans (CS-PGs).
 8. The method of claim 1 in which the hepaticstem cells are obtained from fetal, neonatal, pediatric or adult liver.9. The method of claim 8 in which the liver is human liver.
 10. Themethod of claim 5 in which the laminin is at a concentration betweenabout 0.1 to about 2 μg/cm².
 11. The method of claim 10 in which thelaminin is at a concentration of about 1 μg/cm².
 12. The method of claim3 in which the type III or IV collagens are individually at aconcentration between about 0.1 to about 15 μg/cm².
 13. The method ofclaim 1 in which the layer comprises hyaluronans.
 14. A method ofdifferentiating hepatic stem cells in vitro to hepatoblasts comprising:culturing a population of isolated hepatic stem cells in serum-freeculture media and on a layer of matrix components selected from thegroup consisting of embryonic collagens, basal adhesion molecules,CS-PGs, and combinations thereof, in which the layer is essentially freeof mature collagens, and in which the culturing propagates the hepaticstem cells without inducing their differentiation.
 15. The method ofclaim 14 in which any or all of the matrix components are supplied byactivated endothilia, activated hepatic stellate feeder cells, or both.16. The method of claim 14 in which the embryonic collagen is type IVcollagen.
 17. The method of claim 14 in which the basal adhesionmolecules comprise fetal isoforms of laminin.
 18. The method of claim 14in which the layer further comprises hyaluronans.
 19. The method ofclaim 14 in which the hepatic stem cells are obtained from fetal,neonatal, pediatric or adult liver.
 20. The method of claim 19 in whichthe liver is human liver.
 21. A method of differentiating hepatic stemcells or hepatoblasts in vitro into committed hepatocyte or biliaryprogenitors and progeny thereof comprising: culturing a population ofisolated hepatic stem cells in serum-free culture media and on a layerof matrix components selected from the group consisting of sulfatedproteoglycans, mature collagens, fibronectin, and combinations thereof,and in which the culturing induces the differentiation of the hepaticstem cells or hepatoblasts into committed hepatic or biliary progenitorsand progeny thereof.
 22. The method of claim 21 in which any or all ofthe matrix components are supplied by stromal feeder cells, activatedhepatic stellate feeder cells, myofibroblast feeder cells, orcombinations thereof.
 23. The method of claim 21 in which the maturecollagen is collagen type I.
 24. The method of claim 21 in which thelayer is substantially free of hyaluronans.
 25. The method of claim 21in which the hepatic stem cells are obtained from fetal, neonatal,pediatric or adult liver.
 26. The method of claim 25 in which the liveris human liver.
 27. The method of claim 21 in which the sulfatedproteoglycans are heparan sulfate-PG or heparin-PG, or both.
 27. Acontainer for propagation of hepatic progenitors comprising: (a) acontainer, and (b) a layer of matrix components selected from the groupconsisting of hyaluronans, other unsulfated or poorly sulfatedglycosaminoglycans (GAGs), unsulfated or poorly sulfated proteoglycans,embryonic collagens and embryonic basal adhesion molecules, andcombinations thereof; wherein the layer is essentially free of maturecollagens; and wherein the layer of matrix components substantiallycoats at least one surface of the container.
 29. The container of claim1 in which the container is a tissue culture plate, a bioreactor, a labcell or a lab chip.
 30. A container for propagation of hepaticprogenitors comprising: (a) a container, and (b) a layer of matrixcomponents selected from the group consisting of embryonic collagens,basal adhesion molecules, CS-PGs, and combinations thereof, wherein thelayer is essentially free of mature collagens; and wherein the layer ofmatrix components substantially coats at least one surface of thecontainer.
 31. A container for propagation of hepatic progenitorscomprising: (a) a container, and (b) a layer of matrix componentsselected from the group consisting of sulfated proteoglycans, maturecollagens, fibronectin, and combinations thereof, wherein the layer ofmatrix components substantially coats at least one surface of thecontainer.